Early diagnosis of conformational diseases

ABSTRACT

A method for the diagnosis or detection of conformational diseases by assaying for a marker (the pathogenic conformer) of such diseases in a sample is described, which method comprises a cyclic amplification system to increase the levels of the pathogenic conformer which causes such diseases. In particular, such transmissible conformational diseases may be prion encephalopathies. Assays, diagnostic kits and apparatus based on such methods are also disclosed.

FIELD OF THE INVENTION

The present invention relates to a method for the diagnosis or detectionof conformational diseases by assaying for a marker (i.e. the pathogenicconformer) of such diseases within a sample, which method comprises acyclic amplification system to increase the levels of the pathogenicconformer. In particular, such conformational diseases may be prionencephalopathies.

BACKGROUND OF THE INVENTION

Conformational diseases are a group of disorders apparently unrelated toeach other, but sharing a striking similarity in clinical presentationsthat reflect their shared molecular mechanisms of initiation andself-association, with consequent tissue deposition and damage.

The structural interest is due to the fact that these varied diseaseseach arise from an aberrant conformational transition in an underlingprotein, characteristically leading to protein aggregation and tissuedeposition. Medically, the presentation of these conformational diseasesreflects this molecular mechanism, with typically a slow and insidiousonset when the transition is occurring in a normal protein, but a moresudden onset when it occurs in an unstable variant of the protein. Twoexamples of special significance of such conformational diseases are theTransmissible Spongiform Encephalopathies and Alzheimer dementia, adisease that threatens to overwhelm health care systems in the developedworld (for a review see Carrell et al., 1997).

Transmissible spongiform encephalopathies (TSE) also known as priondiseases are a group of neurodegenerative diseases that affect humansand animals. Creutzfeldt-Jakob disease (CJD), kuru,Gerstmann-Straussler-Scheiker disease (GSS) and fatal familial insomnia(FFI) in humans as well as scrapie and bovine spongiform encephalopathy(BSE) in animals are some of the TSE diseases (Prusiner, 1991).

Although these diseases are relatively rare in humans, the risk for thetransmissibility of BSE to humans through the chain food has taken theattention of the public health authorities and the scientific community(Cousens et al., 1997, Bruce et al., 1997).

These diseases are characterized by an extremely long incubation period,followed by a brief and invariably fatal clinical disease (Roos et al.,1973). To date no therapy is available.

The key characteristic of the disease is the formation of an abnormallyshaped protein named PrP^(Sc), which is a post-translationally modifiedversion of a normal protein, termed PrP^(C) (Cohen and Prusiner, 1998).Chemical differences have not been detected to distinguish between PrPisoforms (Stahl et al., 1993) and the conversion seems to involve aconformational change whereby the α-helical content of the normalprotein diminishes and the amount of β-sheet increases (Pan et al.,1993). The structural changes are followed by alterations in thebiochemical properties: PrP^(C) is soluble in non-denaturing detergents,PrP^(Sc) is insoluble; PrP^(C) is readily digested by proteases, whilePrP^(Sc) is partially resistant, resulting in the formation of aN-terminally truncated fragment known as “PrPres” (Baldwin et al., 1995;Cohen and Prusiner, 1998), “PrP 27-30” (27-30 kDa) or “PK-resistant”(proteinase K resistant) form.

At present there is not an accurate diagnosis for TSE (WHO Report, 1998,Budka et al., 1995, Weber et al., 1997). Attempts to develop adiagnostic test for prion diseases are hampered by the apparent lack ofan immune response to PrP^(Sc). The clinical diagnosis of CJD is basedupon the combination of subacute progressive dementia (less than 2years), myoclonus, and multifocal neurological dysfunction, associatedwith a characteristic periodic electroencephalogram (EEG) (WHO Report,1998, Weber et al., 1997). However, variant CJD (vCJD), most of theiatrogenic forms of CJD and up to 40% of the sporadic cases do not havethe EEG abnormalities (Steinhoff et al., 1996). On average the accuracyof clinical diagnosis is around 60% for CJD and highly variable forother prion-related diseases. The clinical diagnosis is more accurateonly at the late-stage of the disease when clear symptoms have developed(Weber et al., 1997).

Genetic analysis is useful for the diagnosis of inherited priondiseases, but these represent only 15% of the cases. Neuroimaging isuseful only to exclude other conditions of rapidly progressive dementiadue to structural lesions of the brain (Weber et al., 1997). Thefindings obtained by imaging of the brain by computed tomography (CT)and magnetic resonance imaging (MRI) depend mainly on the stage of thedisease. CT is much less sensitive and in early phase no atrophy isdetected in 80% of the cases (Galvez and Cartier, 1983). MRIhyperintense signals have been detected in the basal ganglia besidesatrophy (Onofrji et al., 1993). Like the changes observed by CT, thesealterations are by no means specific.

Recent data have identified several neuronal, astrocytic and glialproteins that are elevated in CJD (Jimi et al., 1992). The proteinS-100, neuron specific isoenzyme and ubiquitin are significantlyincreased in the cerebrospinal fluid (CSF) in the early phase of diseasewith decreasing concentrations over the course of the illness (Jimi etal., 1992). A marker of neuronal death, the 14-3-3 protein, has beenproposed as a specific and sensitive test for sporadic CJD (Hsich etal., 1996). However, it is not useful for the diagnosis of vCJD, andmuch less specific in the genetic forms. As the 14-3-3 protein may bepresent in the CSF of patients with other conditions, the test is notrecommended by WHO as a general screening for CJD and is reserved toconfirm the clinical diagnosis (WHO Report, 1998).

By combining clinical data with the biochemical markers a higher successin the diagnosis is achieved. However, according to the operationaldiagnosis currently in use in the European Surveillance of CJD,definitive diagnosis is established only by neuropathologicalexamination and detection of PrP^(Sc) either by immunohistochemistry,histoblot or western blot (Weber et al., 1997, Budka et al., 1995).

Formation of PrP^(Sc) is not only the most likely cause of the disease,but it is also the best known marker. Detection of PrP^(Sc) in tissuesand cells correlates widely with the disease and with the presence ofTSE infectivity, and treatments that inactivate or eliminate TSEinfectivity also eliminate PrP^(Sc) (Prusiner, 1991). The identificationof PrP^(Sc) in human or animal tissues is considered key for TSEdiagnosis (WHO Report, 1998). One important limitation to this approachis the sensitivity, since the amounts of PrP^(Sc) are high (enough fordetection with conventional methods) only in the CNS at the late stagesof the disease. However, it has been demonstrated that at earlier stagesof the disease there is a generalized distribution of PrP^(Sc) (in lowamounts), especially in the lymphoreticular system (Aguzzi, 1997).Indeed, the presence of PrP^(Sc) has been reported in palatine tonsillartissue and appendix obtained from patients with vCJD (Hill et al.,1997). Although it is not known how early in the disease coursetonsillar or appendix biopsy could be used in vCJD diagnosis, it hasbeen shown that in sheep genetically susceptible to scrapie, PrP^(Sc)could be detected in tonsillar tissue presymptomatically and early inthe incubation period. However, PrP^(Sc) has not been detected in thesetissues so far in any cases of sporadic CJD or GSS (Kawashima et al.,1997).

The normal protein is expressed in white blood cells and platelets andtherefore it is possible that some blood cells may contain PrP^(Sc) inaffected individuals (Aguzzi, 1997). This raises the possibility of ablood test for CJD, but this would require an assay with a much greaterdegree of sensitivity than those currently available.

Prion replication is hypothesized to occur when PrP^(Sc) in theinfecting inoculum interacts specifically with host PrP^(C), catalyzingits conversion to the pathogenic form of the protein (Cohen et al.,1994). This process takes from many months to years to reach aconcentration of PrP^(Sc) enough to trigger the clinical symptoms.

The infective unit of PrP^(Sc) seems to be a β-sheet rich oligomericstructure, which converts the normal protein by integrating it into thegrowing aggregate (FIG. 1). The conversion has been mimicked in vitro bymixing purified PrP^(C) with a 50-fold molar excess of previouslydenatured PrP^(Sc) (Kocisko et al., 1994).

The in vitro conversion systems described so far have low efficiency,since they require an excess of PrP^(Sc) and therefore are not usefulfor diagnostic purposes because they cannot monitor undetectable amountsof the marker. The reason for the low efficiency is that the number ofPrP^(Sc) oligomers (converting units) remains fixed throughout thecourse of the assay. The converting units grow sequentially by the endsand as a result they become larger, but do not increase in number (FIG.1).

DETAILED DESCRIPTION OF THE INVENTION

We have now found a method for the diagnosis or detection of aconformational disease, wherein the disease is characterized by aconformational transition of an underlying protein between anon-pathogenic and a pathogenic conformer, by assaying a marker of saiddisease within a sample, which method comprises:

-   (i) contacting said sample with an amount of the non-pathogenic    conformer;-   (ii) disaggregating any aggregates eventually formed during step    (i); and-   (iii) determining the presence and/or amount of said pathogenic    conformer within the sample.

Generally, the pathogenic conformer will be the marker for the presenceof the said disease.

Preferably, step (i) comprises step (ia) incubating saidsample/non-pathogenic conformer.

According to a preferred embodiment of the invention, steps (ia) and(ii) form a cycle which is repeated at least twice before carrying outstep (iii). More preferably, the cycles are repeated from 5 to 40 times,and most preferably 5-20 times.

The conformational diseases to be detected or diagnosed are those thatare characterised by a conformational transition of an underlyingprotein. This “underlying protein” is a protein which is capable ofadopting a non-pathogenic conformation and a pathogenic conformation.One example of such a protein is the prion protein, PrP. A furtherexample of such a protein is the protein involved in Alzheimer'sdisease, i.e. the β-amyloid protein.

The conformational diseases to be diagnosed or detected are preferablytransmissible conformational diseases, such as TSE (as defined in theBackground section).

In the case of diagnosis of TSE and according to a preferred embodimentof the invention, the marker of the disease as well as the pathogenicconformer is PrP^(Sc), whereas the non-pathogenic conformer of theprotein of interest is PrP^(C).

The amount of the non-pathogenic conformer that is used in step (i) (andoptionally in step (ib)) will generally be a known amount, although thisneed not be the case if one merely wishes to establish the presence orabsence of the pathogenic conformer.

Preferably, the amount of non-pathogenic conformer that is used in step(i) (and optionally in step (ib)) will be an excess amount. Generally,the initial ratio of non-pathogenic conformer to pathogenic conformer(if present in the sample) will be greater than 100:1, preferablygreater than 1000:1 and most preferably greater than 1000000:1.

In a further preferred embodiment of the invention, the non-pathogenicconformer in step (i) is present in a brain homogenate of a healthysubject and/or may be added to it, before carrying out step (i); in thiscase, therefore, the brain homogenate containing a (preferably known)excess of the non-pathogenic conformer is added during step (i).Preferably, the brain homogenate of the healthy subject comes from thesame species from which the sample to be analyzed comes (e.g. humanbrain homogenate for human sample to be analyzed, rat brain homogenatefrom rat sample to be analyzed). More preferably, the non-pathogenicconformer is present in a specific fraction of the brain homogenate, forexample in the lipid-rafts from brain homogenate. The preparation ofsuch fractions can be carried out for example as described in SargiacomoM et al., 1993.

Thus the invention further relates to a method or assay as describedherein wherein a tissue or tissue fraction is added to thenon-pathogenic conformer in step (i). Preferably, the tissue is braintissue, or a homogenate or fraction derived therefrom, from a healthysubject (i.e. one where the pathogenic conformer is not present).

It has been reported (Kocisko et al., 1994) that less glycosylated formsof PrP^(C) are preferentially converted to the PrP^(Sc) form. Inparticular, PrP^(C) which was treated with phosphatidylinositol specificphospholipase C was routinely more efficiently converted to thepathogenic form than the complete, more heavily glycosylated PrP^(C). Afurther embodiment of the invention therefore relates to a method orassay as herein described wherein the non-pathogenic conformer isPrP^(C) which has a reduced level of glycosylation (in particularN-linked glycosylation) in comparison with the wild-type PrP^(C).Preferably, the PrP^(C) has been treated to remove some, all or asignificant amount of the glycosylation prior to its use as thenon-pathogenic conformer in the methods and assays described herein; andmore preferably, the non-pathogenic conformer is PrP^(C) which isessentially unglycosylated.

In the case of diagnosis of TSE, if aggregates of the pathogenic formare present within the sample, during step (i) they will induce thePrP^(C)→PrP^(Sc) transition and during step (ii) such aggregates will bebroken down into smaller still infective units, each of which is stillcapable of inducing the conversion of other PrP^(C). This kind of methodis herein called “cyclic amplification” and is represented in FIG. 2.This system results in an exponential increase in the amount of PrP^(Sc)eventually present in the sample that can easily be detected. Accordingto a further preferred embodiment of the invention, it is thereforepossible to calculate the amount of PrP^(Sc) initially present in thesample starting from the known amount of PrP^(C), determining the amountof PrP^(Sc) present within the sample at the end of the assay andconsidering the number of cycles performed.

If, on the contrary, no PrP^(Sc) (either as such or in the form ofaggregates) is present in the sample, no PrP^(C) molecule will beconverted into PrP^(Sc) and at the end of the assay the marker will becompletely absent (no pathogenic conformer detected in the sample).

It has been shown that the infective unit of PrP^(Sc) is a β-sheet richoligomer, which can convert the normal protein by integrating it intothe growing aggregate, where it acquires the properties associated withthe abnormal form (protease resistance and insolubility) (Jarrett andLansbury, Jr., 1993, Caughey et al., 1997). After incubation of the twoforms of PrP, the oligomeric species increases its size by recruitingand transforming PrP^(C) molecules. This process has low efficiency,since it depends on a fixed number of oligomers growing by the ends. Thenumber of converting units is not increased in the course of thereaction when they only become larger. It is assumed that this processis what happens in the animal or human body after infection; a processknown to take months or even several years. In this invention wedescribe a procedure to break down the oligomers to a smaller ones, eachof which is then capable of converting PrP^(C).

Therefore, the system has direct applications to the diagnosis ofconformational diseases, and in particular transmissible conformationaldiseases, such as TSE by amplifying otherwise undetectable amounts ofPrP^(Sc) in different tissues or biological fluids. The system may allowthe early identification of people at risk of developing TSE and couldalso be very useful to follow biochemically the efficacy of TSEtherapeutic compounds during clinical trials.

According to a preferred embodiment of the invention the sample to beanalysed is subjected to a “pre-treatment” step, which has the purposeof “selectively concentrating” in the sample the pathogenic conformerthat is to be detected. In the case of TSE both PrP^(C) and PrP^(Sc)have been reported to be located in a special region of the plasmamembrane which is resistant to mild detergent treatment (such asice-cold Triton X-100) due to the relatively high content of cholesteroland glycosphingolipids (M. Vey et al., 1996). These membrane domains arenamed lipid-rafts or detergent-resistant membranes (DRM) orcaveolae-like domains (CLDs) and are rich in signaling proteins,receptors and GPI-anchored proteins. We have confirmed that 100% ofPrP^(C) in brain is attached to this fraction, which contains <2% of thetotal proteins (see Example 6 and FIG. 7). Thus, the simple step oflipid-raft isolation from the sample allows a dramatic enrichment inPrP^(C). Similar results were obtained by the Applicant in the isolationof lipid-rafts from scrapie brain homogenate, in which PrP^(Sc) wasrecovered in the rafts.

Thus one embodiment of the invention includes a step wherein the sampleto be analysed is subjected to a pre-treatment step for selectivelyconcentrating the pathogenic conformer in the sample. Preferably, thepathogenic conformer is PrP^(Sc) and the pretreatment is the extractionfrom the sample of a fraction which is insoluble in mild detergents.

Steps (i) and (ia) are preferably performed under physiologicalconditions (pH, temperature and ionic strength) and, more preferably,protease inhibitors and detergents are also added to the solution. Theconditions will be chosen so as to allow any pathogenic conformer, ifpresent in the sample, to convert the non-pathogenic conformer intopathogenic conformer thus forming an aggregate or oligomer of pathogenicconformers. Appropriate physiological conditions will readily beapparent to those skilled in the art.

The length of the incubation will be for a time which will allow some,all or a significant portion of the non-pathogenic conformer to beconverted to pathogenic conformer, assuming that the sample containssome pathogenic conformer. The time will readily be determinable bythose skilled in the art. Preferably, each incubation will be between 1minute to 4 hours, most preferably 30 minutes to 1 hour, andparticularly preferably approximately 60 minutes.

Incubation step (ia) may also comprise the further step (ib) whichcomprises the addition of a further amount of non-pathogenic conformer.

Various methods can be used for disaggregating the aggregates duringstep (ii) of the method of the present invention. They include:treatment with solvents (such as sodium dodecyl sulfate,dimethylsulfoxide, acetonitrile, guanidine, urea, trifluoroethanol,diluted trifluroacetic acid, diluted formic acid, etc.), modification ofthe chemical-physical characteristics of the solution such as pH,temperature, ionic strength, dielectric constant, and physical methods,such as sonication, laser irradiation, freezing/thawing, French press,autoclave incubation, high pressure, stirring, mild homogenization,other kinds of irradiation, etc. Sonication is the preferred methodaccording to the invention

Disaggregation may be carried out for a time which disaggregates some,all or a significant portion of the aggregates which have formed duringstep (ii). It is not necessary for all of the aggregates to bedisaggregated in any one disaggregation step. In this way, the number ofconverting units is increased in each disaggregation step.

The disaggregation time will readily be determinable by those skilled inthe art and it may depend on the method of disaggregation used.Preferably, disaggregation is carried out for 1 second to 60 minutes,most preferably 5 seconds to 30 minutes and particularly preferably, 5seconds to 10 minutes. If disaggregation is carried out by sonication,sonication is preferably for 5 seconds to 5 minutes, and most preferablyfor 5 to 30 seconds.

Sonication has been used in the past as part of several methods topurify PrP with the goal of increasing solubility of large aggregates,but it has never been described to amplify in vitro conversion of PrP.

The use of a traditional single-probe sonicator imposes a problem forhandling many samples simultaneously, such as a diagnostic test willrequire. There are on the market some 96-well format microplatesonicators, which provide sonication to all the wells at the same timeand can be programmed for automatic operation. These sonicators can beeasily adapted to be used in the diagnostic method of the presentinvention.

Thus one embodiment of the invention relates to the use, in step (ii),of a multi-well sonicator.

The detection of the newly converted pathogenic conformer, e.g.PrP^(Sc), (iii) after the cyclic amplification procedure described insteps (i) to (ii) could be carried out according to any of the knownmethods. Specific detection of PrP^(Sc) is usually (but not always, seebelow) done by a first step of separation of the two PrP isoforms(normal protein and pathogenic protein). Separation is done on the basisof the peculiar biochemical properties of PrP^(Sc) that distinguish itfrom most of the normal proteins of the body, namely: PrP^(Sc) ispartially resistant to protease treatment and is insoluble even in thepresence of non-denaturant detergents. Therefore the first step afterthe amplification procedure is usually the removal or separation ofPrP^(C) in the sample, either by treatment with proteases or bycentrifugation to separate the soluble (PrP^(C)) from the insoluble(PrP^(Sc)) protein. Thereafter, detection of PrP^(Sc) can be done by anyof the following methods, inter alia:

A) Immunobloting after SDS-PAGE. This is done through a routineprocedure well known for those with skill in the art and using some ofthe many commercially available anti-PrP antibodies.

B) Elisa assay. Solid phase detection can be done by either a simpleassay in which the sample is loaded on the plate and the amount ofPrP^(Sc) detected afterwards by using anti-PrP antibodies or morepreferably by using sandwich Elisa in which the plate is first coatedwith an anti-PrP antibody that captures specifically PrP from thesample, which is finally detected by using a second anti-PrP antibody.Both forms of Elisa can also be used with labelled (radioactivity,fluorescence, biotin, etc) anti-PrP antibodies to further increase thesensitivity of the detection.

C) Radioactivity assays. Normal PrP^(C) used as a substrate for theamplification procedure can be radioactively labelled (3H, 14C, 35S,125I, etc) before starting the procedure and after the removal of thenon-converted PrP^(C), radioactivity of newly converted PrP^(Sc) couldbe quantitated. This procedure is more quantitative and does not rely onthe use antibodies.

D) Fluorescence assays. Normal PrP^(C) used as a substrate for theamplification procedure can be labelled with fluorescent probes beforestarting the procedure and after the removal of non-converted PrP^(C),fluorescence of the newly converted PrP^(Sc) could be quantitated. It ispossible that the fluorescence assay might not require the removal ofnon-converted PrP^(C), because the fluorescence properties of PrP^(C)and PrP^(Sc) might be different due to the distinct conformation of thetwo isoforms.

E) Aggregation assays. It is well known that PrP^(Sc) (and not PrP^(C))is able to aggregate forming amyloid fibrils or rod-type structures.Therefore detection of PrP^(Sc) could be done by using the methods usedto quantify the formation of these type of aggregates, includingelectron microscopy, staining with specific dyes (Congo red, ThioflavinS and T, etc), and turbidimetric assays. Aggregation assays do notrequire the step of separation of the two isoforms, because is knownthat normal PrPC does not aggregate.

F) Structural assays. The most important difference between the normaland the pathogenic PrP is their secondary and tertiary structures.Therefore, methods that allow the structural evaluation of proteins canbe used, including NMR, Circular dichroism, Fourier-transformed infraredspectroscopy, Raman spectroscopy, intrinsic fluorescence, UV absorption,etc.

The most widely used PrP monoclonal antibody is “3F4” (Kascsak et al.,1987), which is a monoclonal antibody derived from a mouse immunizedwith hamster 263K PrPres (the protease-resistant conformer). Thisantibody is also able to recognize the non-pathogenic conformer fromhamsters and humans, but not from bovine, mouse, rat, sheep or rabbitbrains; it is also able to bind the human pathogenic conformer, but onlyafter denaturation of the protein.

Such antibodies may be labeled to allow easy detection of the marker.For example time-resolved fluorescence measurements witheuropium-labeled 3F4 antibody has been used by some scientists (Safar etal., 1998).

The above-described methods of detection may be used for the detectionof other pathogenic conformers, for example the pathogenic form ofβ-amyloid protein, mutatis mutandis.

In an alternative embodiment the non-pathogenic conformer added inexcess may be labeled and detectable so that the amount of thenon-aggregated conformer at the end of the assay will allow adetermination of the amount of pathogenic conformer initially present inthe sample.

According to a further alternative embodiment, the pathogenic conformer(the marker) could be directly detected with an antibody directedagainst it.

In broader terms a label or labeling moiety may be added to thepathogenic conformer, to the non-pathogenic conformer or to an antibodyagainst one of the conformers depending on the kind of assay that isperformed.

Another object of the invention is an assay for a marker of aconformational disease which is characterized by a conformationaltransition of an underlying protein between a non-pathogenic and apathogenic conformer, within a sample, which assay comprises thefollowing steps: (i) contacting said sample with an amount of thenon-pathogenic conformer, (ii) disaggregating any aggregates eventuallyformed during step (i) and (iii) determining the presence and/or amountof said pathogenic conformer within the sample. In general, thepathogenic conformer will be the marker for the presence of saiddisease.

Preferably, step (i) comprises step (ia) incubating saidsample/non-pathogenic conformer.

According to a preferred embodiment of the invention, steps (ia) and(ii) form a cycle which is repeated at least twice before carrying outstep (iii). More preferably, the cycles are repeated from 5 to 40 times,and most preferably 5 to 20 times.

A further object of the present invention is a diagnostic kit for use inthe assay specified, which comprises an amount of the non-pathogenicconformer, and optionally additionally a micro-titre plate and amulti-well sonicator.

Using the method of the invention, it is possible to detect 1 to 10 fgof pathogenic conformer initially present in a sample, which isequivalent to 3 to 30×10⁻²⁰ moles.

The sample will generally be a biological sample or tissue, and any suchbiological sample or tissue can be assayed with the method of thepresent invention. In the case of a tissue, the assay and method of thepresent invention may be carried out on homogenates or direct on ex vivosamples. The methods and assays will generally be carried out on ex vivoor in vitro samples. Preferably, the sample is a biological fluid, suchas blood, lymph, urine or milk; brain tissue, spinal cord, tonsillartissue or appendix tissue; a sample derived from blood such as bloodcell ghosts or buffy coat preparations; or a plasma membrane preparationsuch as lipid-rafts, detergent resistant membranes or caveolae-likedomains. The sample might alternatively be a composition comprising acompound (particularly a protein) derived from a human or animal source,such as growth hormone, or a tissue extract, such as pituitary extract.Such a sample composition might be contaminated with a pathogenicconformer.

The sample might also comprise a food product or drink, or a portion ofa food product or drink (either destined for human consumption or animalconsumption) in order to establish the presence or absence of pathogenicconformer in that product or drink.

Preferably, the non-pathogenic conformer added in step (i) will be fromthe same species as the sample. It may, for example, be derived from ahealthy (i.e. non-pathogenic) form (e.g. tissue) of the biologicalsample to be tested. Alternatively, the non-pathogenic conformer may beproduced synthetically or recombinantly, using means known in the art.It will be understood, however, that the non-pathogenic conformer neednot be in pure or even substantially pure form. In most cases, thenon-pathogenic conformer will be in the form of a tissue homogenate or afraction thereof which contains the relevant non-pathogenic conformer.Preferred examples include brain homogenates and fractions derivedtherefrom, e.g. lipid rafts.

Preferably, the sample and/or the non-pathogenic conformer will be ofhuman origin or from a domestic animal, e.g. a cow, sheep, goat or cat.

Another object of the present invention is to provide a method foridentifying a compound which modulates the conformational transition ofan underlying protein between a non-pathogenic and a pathogenicconformer, comprising:

-   (i) contacting an amount of the non-pathogenic conformer with an    amount of the pathogenic conformer in the presence and in the    absence of said compound,-   (ii) disaggregating any aggregates eventually formed during step    (i),-   (iii) determining the amount of the pathogenic conformer in the    presence and in the absence of said compound.

If desired, step (i) may comprise step (ia) incubating saidsample/non-pathogenic conformer, and a cycle carried out between steps(ia) and (ii) as described above for the methods and assays of theinvention, mutatis mutandis.

If the amount of pathogenic conformer measured in the presence of thecompound is higher than that measured in the absence, it means that thecompound is a factor which “catalyzes” the conformational transition; ifsuch amount is lower, it means that the compound is a factor whichinhibits such transition.

According to the above method, “identifying” should also be interpretedto mean “screening” of a series of compounds.

A “label” or “labelling moiety” may be any compound employed as a meansfor detecting a protein. The label or labelling moiety may be attachedto the protein via ionic or covalent interactions, hydrogen bonding,electrostatic interactions or intercalation. Examples of labels andlabelling moieties include, but are not limited to fluorescent dyeconjugates, biotin, digoxigenin, radionucleotides, chemiluminescentsubstances, enzymes and receptors, such that detection of the labelledprotein is by fluorescence, conjugation to streptaviden and/or avidin,quantitation of radioactivity or chemiluminescence, catalytic and/orligand-receptor interactions. Preferably it is a fluorescent or aphosphorescent label.

The term “conformational diseases” refers to that group of disordersarising from a propagation of an aberrant conformational transition ofan underlying protein, leading to protein aggregation and tissuedeposition. Such diseases can also be transmitted by an inducedconformational change, propagated from a pathogenic conformer to itsnormal or non-pathogenic conformer and in this case they are calledherein “transmissible conformational diseases”. Examples of such kindsof diseases are the prion encephalopathies, including the bovinespongiform encephalopathy (BSE) and its human equivalentCreutzfeld-Jakob (CJD) disease, in which the underlying protein is thePrP.

The term “sporadic CJD” abbreviated as “sCJD” refers to the most commonmanifestation of Creutzfeldt-Jakob Disease (CJD). This disease occursspontaneously in individuals with a mean age of approximately 60 at arate of 1 per million per year individuals across the earth.

The term “Iaterogenic CJD” abbreviated as “iCJD” refers to diseaseresulting from accidental infection of people with human prions. Themost noted example of such is the accidental infection of children withhuman prions from contaminated preparations of human growth hormone.

The term “Familial CJD” refers to a form of CJD, which occurs rarely infamilies and is inevitably caused by mutations of the human prionprotein gene. The disease results from an autosomal dominant disorder.Family members who inherit the mutations succumb to CJD.

The term “Gerstmann-Strassler-Scheinker Disease” abbreviated as “GSS”refers to a form of inherited human prion disease. The disease occursfrom an autosomal dominant disorder. Family members who inherit themutant gene succumb to GSS.

The term “prion” shall mean a transmissible particle known to cause agroup of such transmissible conformational diseases (spongiformencephalopathies) in humans and animals. The term “prion” is acontraction of the words “protein” and “infection” and the particles arecomprised largely if not exclusively of PrP^(Sc) molecules.

Prions are distinct from bacteria, viruses and viroids. Known prionsinclude those which infect animals to cause scrapie, a transmissible,degenerative disease of the nervous system of sheep and goats as well asbovine spongiform encephalopathies (BSE) or mad cow disease and felinespongiform encephalopathies of cats. Four prion diseases known to affecthumans are (1) kuru, (2) Creutzfeldt-Jakob Disease (CJD), (3)Gerstmann-Strassler-Scheinker Disease (GSS), and (4) fatal familialinsomnia (FFI). As used herein prion includes all forms of prionscausing all or any of these diseases or others in any animals used andin particular in humans and in domesticated farm animals.

The terms “PrP gene” and “prion protein gene” are used interchangeablyherein to describe genetic material which expresses the prion proteinsand polymorphisms and mutations such as those listed herein under thesubheading “Pathogenic Mutations and Polymorphisms.” The PrP gene can befrom any animal including the “host” and “test” animals described hereinand any and all polymorphisms and mutations thereof, it being recognizedthat the terms include other such PrP genes that are yet to bediscovered.

The term “PrP gene” refers generally to any gene of any species whichencodes any form of a PrP amino acid sequences including any prionprotein. Some commonly known PrP sequences are described in Gabriel etal., 1992, which is incorporated herein by reference to disclose anddescribe such sequences.

Abbreviations used herein include:

-   CNS for central nervous system;-   BSE for bovine spongiform encephalopathy;-   CJD for Creutzfeldt-Jakob Disease;-   FFI for fatal familial insomnia;-   GSS for Gerstmann-Strassler-Scheinker Disease;-   PrP for prion protein;-   PrP^(C) for the normal, non-pathogenic conformer of PrP;-   PrP^(Sc) for the pathogenic or “scrapie” isoform of PrP (which is    also the marker for prion diseases).    Pathogenic Mutations and Polymorphisms

There are a number of known pathogenic mutations in the human PrP gene.Further, there are known polymorphisms in the human, sheep and bovinePrP genes.

The following is a non-limiting list of such mutations andpolymorphisms:

MUTATION TABLE Pathogenic human Human Sheep Bovine mutationspolymorphisms polymorphisms polymorphisms 2 octarepeat Codon 129 Codon171 5 or 6 insert Met/Val Arg/Glu octarepeats 4 octarepeat Codon 219Codon 136 insert Glu/Lys Ala/Val 5 octarepeat insert 6 octarepeat insert7 octarepeat insert 8 octarepeat insert 9 octarepeat insert Codon 102Pro-Len Codon 105 Pro-Leu Codon 117 Ala-Val Codon 145 Stop Codon 178Asp-Asn Codon 180 Val-Ile Codon 198 Phe-Ser Codon 200 Glu-Lys Codon 210Val-Ile Codon 217 Asn-Arg Codon 232 Met-Ala

The normal amino acid sequence, which occurs in the vast majority ofindividuals, is referred to as the wild-type PrP sequence. Thiswild-type sequence is subject to certain characteristic polymorphicvariations. In the case of human PrP, two polymorphic amino acids occurat residues 129 (Met/Val) and 219 (Glu/Lys). Sheep PrP has two aminoacid polymorphisms at residues 171 and 136, while bovine PrP has eitherfive or six repeats of an eight amino acid motif sequence in the aminoterminal region of the mature prion protein. While none of thesepolymorphisms are of themselves pathogenic, they appear to influenceprion diseases. Distinct from these normal variations of the wild-typeprion proteins, certain mutations of the human PrP gene which altereither specific amino acid residues of PrP or the number of octarepeatshave been identified which segregate with inherited human priondiseases.

In order to provide further meaning to the above chart demonstrating themutations and polymorphisms, one can refer to the published sequences ofPrP genes. For example, a chicken, bovine, sheep, rat and mouse PrP geneare disclosed and published within Gabriel et al., 1992. The sequencefor the Syrian hamster is published in Baslet et al 1986. The PrP geneof sheep is published by Goldmann et al., 1990. The PrP gene sequencefor bovine is published in Goldmann et al., 1991. The sequence forchicken PrP gene is published in Harris et al., 1991. The PrP genesequence for mink is published in Kretzschmar et al., 1992. The humanPrP gene sequence is published in Kretzschmar et al., 1986. The PrP genesequence for mouse is published in Locht et al., 1986. The PrP genesequence for sheep is published in Westaway et al., 1994. Thesepublications are all incorporated herein by reference to disclose anddescribe the PrP gene and PrP amino acid sequence.

The invention also provides a method for detecting the presence of apathogenic form of prion protein within a sample (preferably a blood orbrain sample) comprising:

-   (i) contacting the sample with an amount of non-pathogenic prion    protein;-   (ia) incubating the sample/non-pathogenic prion protein;-   (ii) disaggregating any aggregates formed during step (ia);

repeating steps (ia)-(ii) two or more times; and then

-   (iii) determining the presence and/or amount of pathogenic prion    protein within the sample.

A further embodiment of the invention provides a method for diagnosingCJD within a patient, comprising: taking a sample from the patient(preferably a blood or brain sample);

-   (i) contacting the sample with an amount of PrP^(C) protein;-   (ia) incubating the sample/PrP^(C) protein;-   (ii) disaggregating any aggregates formed during step (ia);

repeating steps (ia)-(ii) two or more times; and then

-   (iii) determining the presence and/or amount of PrP^(Sc) within the    sample.

The invention also provides a method for detecting the presence of apathogenic form of β-amyloid protein within a sample (preferably a bloodor brain sample), comprising:

-   (i) contacting the sample with an amount of non-pathogenic β-amyloid    protein;-   (ia) incubating the sample/non-pathogenic β-amyloid protein;-   (ii) disaggregating any aggregates formed during step (ia);

repeating steps (ia)-(ii) two or more times; and then

-   (iii) determining the presence and/or amount of pathogenic β-amyloid    protein within the sample.

A further embodiment of the invention provides a method for diagnosingAlzheimer's disease in a patient, comprising:

-   taking a sample (preferably a blood or brain sample) from the    patient;-   (i) contacting the sample with an amount of non-pathogenic β-amyloid    protein;-   (ia) incubating the sample/non-pathogenic β-amyloid protein;-   (ii) disaggregating any aggregates formed during step (ia);

repeating steps (ia)-(ii) two or more times; and then

-   (iii) determining the presence and/or amount of pathogenic β-amyloid    protein within the sample.

The invention furthermore provides apparatus for use in the methodsdescribed above, particularly apparatus comprising a microtitre plate,multi-well sonicator and an amount of a non-pathogenic conformer.

A further embodiment of the invention provides a method for thediagnostic detection of a conformational disease, characterized by aconformational transition of an underlying protein between anon-pathogenic and a pathogenic conformer, by assaying a marker of saiddisease within a sample, which method comprises (i) contacting saidsample with a known amount of the non-pathogenic conformer, (ii)disaggregating the aggregates eventually formed during step (i) and(iii) determining the presence and/or amount of said pathogenicconformer within the sample. Preferably, steps (i) and (ii) form a cyclewhich is repeated at least twice before carrying out step (iii), mostpreferably steps (i) and (ii) form a cycle, which is repeated from 5 to40 times before carrying out step (iii).

The invention also provides an assay for a marker of a conformationaldisease, characterized by a conformational transition of an underlyingprotein between a non-pathogenic and a pathogenic conformer, within asample, which assay comprises the following steps: (i) contacting saidsample with a known amount of the non-pathogenic conformer, (ii)disaggregating the aggregates eventually formed during step (i) and(iii) determining the presence and/or amount of said pathogenicconformer within the sample. Preferably, the steps (i) and (ii) form acycle which is repeated at least twice before carrying out step (iii).

The invention further provides a method for identifying a compound whichmodulates the conformational transition of an underlying protein betweena non-pathogenic and a pathogenic conformer, comprising:

-   (i) contacting a known amount of the non-pathogenic conformer with a    known amount of the pathogenic conformer in the presence and in the    absence of said compound,-   (ii) disaggregating the aggregates eventually formed during step    (i),-   (iii) determining the amount of the pathogenic conformer in the    presence and in the absence of said compound.

The present invention has been described with reference to the specificembodiments, but the content of the description comprises allmodifications and substitutions, which can be brought by a personskilled in the art without extending beyond the meaning and purpose ofthe claims.

The invention will now be described by means of the following Examples,which should not be construed as in any way limiting the presentinvention. The Examples will refer to the Figures specified here below.

DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of the conversion PrP^(C)→PrP^(Sc). Theinfective unit of PrP^(Sc) is a β-sheet rich oligomer, which convertsPrP^(C) by integrating it into the growing aggregate, where it acquiresthe properties associated with PrP^(Sc).

FIG. 2. Diagrammatic representation of the cyclic amplificationprocedure. The system is based on cycles of incubation of PrP^(Sc) inthe presence of excess of PrP^(C) followed by cycles of sonication.During the incubation periods, oligomeric PrP^(Sc) is enlarged byincorporating PrP^(C) into the growing aggregate, while duringsonication the aggregates are sonication/incubation are shown.

FIG. 3. Amplification of PrP^(Sc) by sonication cycles. A small amountof scrapie brain homogenate containing PrP^(Sc) was incubated withhealthy rat brain homogenate (lane 1, control experiment) or withhealthy hamster brain homogenate (lane 2 and 3). The latter sample wasdivided in two groups one of which was subjected to five cycles ofincubation/sonication (lane 3). Half of the above samples were loadeddirectly in a gel and stained for total protein with Coomasie (panel A).The other half were treated with PK and immunoblotted using the anti-PrPantibody 3F4 (panel B). Panel C shows some controls in which healthybrain homogenate was incubated alone (lanes 1 and 2) or in the presenceof diluted scrapie brain homogenate (lanes 3 and 4). Half of the samples(lanes 2 and 4) were subjected to 5 cycles of sonication/incubation.Lanes 2, 3 and 4 were treated with proteinase K.

FIG. 4. Sensitivity of the cyclic amplification system. The minimumconcentration of PrP^(Sc) that can be used for detection afteramplification was studied by serially diluting the scrapie brainhomogenate and incubating with healthy hamster brain homogenate with orwithout sonication cycles. Panel A shows the control experiment in whichscrapie hamster brain was diluted serially in rat brain homogenate.Panel B corresponds to the experiment in which the serial dilutions ofscrapie hamster brain were incubated with healthy hamster brain andsubjected to 5 cycles of incubation/sonication. Densitometric evaluationof the immunoblots in A and B is shown in panel C. The dilutions weredone considering as starting material the brain and were the following:100 (lane 1), 200 (lane 2), 400 (lane 3), 800 (lane 4), 1600 (lane 5)and 3200 (lane 6).

FIG. 5. Relationship between the PrPres signal and the number ofamplification cycles. Diluted scrapie brain homogenate was incubatedwith an excess of healthy hamster brain homogenate. Samples weresubjected to 0, 5, 10, 20 or 40 cycles and the PrPres signal evaluatedby immunoblot.

FIG. 6. Amplification of PrP^(Sc) in blood samples. Heparinized ratblood was spiked with Scrapie hamster brain homogenate to reach a finaldilution of 10:1. This mixture was incubated for 15 min at RT. 10 foldserial dilutions were made of this material using heparinized rat blood.Samples were subjected to 11 cycles of incubation-sonication and thePrPres signal evaluated by immunoblot.

FIG. 7: Prion protein is present in lipid-rafts. Lipid-rafts (alsocalled detergent-resistant membrane fraction or DRM) were isolated usinga modification of previouly described protocols. One-hundred mg of braintissue was homogenized in 1 ml of PBS containing 1% triton X-100 and 1×complete cocktail of protease inhibitors (Boehringer). Tissue washomogenized with 10 passages through 22 G syringe needle and incubatedfor 30 minutes at 4° C. on a rotary shaker. The sample was diluted 1:2in sucrose 60% and placed in the bottom of a centrifuge tube. 7 ml ofsucrose 35% were place carefully over the sample. 1.5 ml of sucrose 15%was layered in the top of the gradient. The tube was centrifuged at150,000 g for 18 hrs at 4° C. The lipid rafts float to the 15%-35%sucrose interface (panel A). Different fractions were collected andanalyzed by total protein staining with silver nitrate (panel B) andimmunoblot to detect PrP (panel C). To remove sucrose from the sample,lipid raft fraction was recovered washed in PBS and centrifuged at28,000 rpm during 1 hr at 4° C. The pellet was washed and resuspended inPBS containing 0.5% Triton X-100, 0.5% SDS and protease inhibitors. AllPrPC was located in this fraction (panel D).

FIG. 8: The factors needed for amplification are present in lipid-rafts.Lipid-rafts were isolated from healthy hamster brain as describe in FIG.2 and mixed with 700-fold diluted PrPSc highly purified from scrapiehamster brain. Samples were either frozen (line 3) or amplified for 20 h(line 4). Lines 1 and 2 represent the same procedures but using totalbrain homogenate for amplification.

FIG. 9: Presymptomatic detection of PrPSc in hamster brain. Hamsterswere inoculated intra-cerebrally (i.c.) with saline (control group) orwith 100-fold diluted scrapie brain homogenate. Every week 4 hamstersper group were sacrificed and brains were extracted and homogenized.Half of the samples were frozen immediately (white bars) and the otherhalf subjected to 20 cycles of incubation/sonication (black bars). Allsamples were treated with PK and immunoblotted. The intensity of thebands was evaluated by densitometry. Each bar represent the average ofsamples from 4 animals. No detection was observed in any of the controlbrains either without or with amplification and these results are notshown in the Figure.

FIG. 10: Amplification of human PrPSc. The studies were done using brainsamples of 11 different confirmed cases of sporadic CJD, as well as 5from familial CJD and 4 age-matched controls, which included patientsaffected by other neurological disorders. Brain was homogenized andsubjected to 20 amplification samples. Representative results of acontrol (A) and three different sporadic CJD (B) cases (1, 2, 3) areshown in the Figure.

FIG. 11: Detection of PrPSc in blood after preparation of blood cellsghosts. Cell ghosts from 0.5 ml of heparinized blood coming from healthy(C) and scrapie-affected hamsters (Sc) were prepared as described in thetext. Half of the samples were not subjected to amplification and theother half were mixed with normal hamster brain homogenate and subjectedto 20 amplification cycles. All samples were then treated with PK andanalyzed by immunoblots. One representative experiment is shown in theFigure.

FIG. 12: Detection of PrPSc in blood after sarkosyl extraction. 0.5 mlof heparinized blood coming from healthy (C) and scrapie-affectedhamsters (Sc) was subjected to sarkosyl extraction as described in thetext. Half of the samples were not subjected to amplification and theother half were mixed with normal hamster brain homogenate and subjectedto 20 amplification cycles. All samples were then treated with PK andanalyzed by immunoblots. One representative sample of control animalsand two for scrapie-affected animals is shown in the Figure.

FIG. 13: Detection of PrPSc in blood after lipid rafts purification.Lipid-rafts were extracted as described in the text from 0.5 ml ofheparinized blood coming from healthy (C) and scrapie-affected hamsters(Sc). Half of the samples were not subjected to amplification and theother half were mixed with normal hamster brain homogenate and subjectedto 20 amplification cycles. All samples were then treated with PK andanalyzed by immunoblots. One representative sample of control animalsand two for scrapie-affected animals is shown in the Figure.

FIG. 14: Detection of PrPSc in blood after preparation of buffy coats.The buffy coat fraction of blood was separated by centrifugation from0.5 ml of heparinized blood coming from healthy (C) and scrapie-affectedhamsters (Sc). Half of the samples were not subjected to amplificationand the other half were mixed with normal hamster brain homogenate andsubjected to 20 amplification cycles. All samples were then treated withPK and analyzed by immunoblots. One representative experiment is shownin the Figure.

EXAMPLES Example 1 Amplification of PK Resistant PrP by Cyclic in vitroConversion

Hamster brain homogenate extracted from scrapie affected animals wasdiluted until the signal of PrP^(Sc) was barely detected by immunoblotafter treatment with proteinase K (PK) FIG. 3B, lane 1). PK treatment isdone routinely in the field to distinguish between the normal andabnormal forms of PrP, which differ in their sensitivity to proteasedegradation (PrP^(Sc) is partially resistant and PrP^(C) is degraded)(Prusiner, 1991). The form of PrP that is resistant to PK treatment willbe named from now on PrPres. Incubation of a sample of diluted scrapiebrain homogenate with a healthy hamster brain homogenate containing anexcess of PrP^(C), resulted in the increase in PrPres signal (FIG. 3B,lane 2).

This suggests that the incubation of the two brain homogenates resultedin the conversion of PrP^(C) to PrP^(Sc). When the samples wereincubated under the same conditions but subjected to five cycles ofincubation/sonication, the amount of PrPres was dramatically increased(FIG. 3B, lane 3). Densitometric analysis of the immunoblot indicatesthat the PrPres signal was increased 84-fold by cyclic amplification incomparison with the PrPres signal presented in the diluted scrapie brainhomogenate (lane 1).

The conversion is dependent of the presence of PrP^(Sc) since no PrPreswas observed when the normal hamster brain homogenate was incubatedalone under the same conditions either with or without sonication (FIG.3C, lane 2). To rule out artifacts of the transfer, the total proteinloaded in the gel was maintained constant (FIG. 3A) by adding rat brainhomogenate to the diluted scrapie sample, taking advantage of the factthat rat PrP is not detected by the antibody used for the immunoblot.

Example 2 Sensitivity of Detection by Cyclic Amplification

To evaluate the minimum concentration of PrP^(Sc) that can be used fordetection after amplification, the scrapie brain homogenate was seriallydiluted directly in healthy hamster brain homogenate. Withoutincubation, the signal of PrPres diminishes progressively until it wascompletely undetectable at 800-fold dilution (FIGS. 4A, C). In contrastwhen the same dilution was incubated with healthy hamster brainhomogenate and subjected to 5 cycles of incubation/sonication, the limitof PrPres detection was decreased dramatically. Indeed, clear signal waseasily detected even at a 3200-fold dilution (FIGS. 4B, C).

Example 3 Exponential Increase in PrPres with Number of Cycles

To study whether the intensity of the PrPres signal after cyclicamplification depends on the number of cycles of incubation/sonicationperformed, diluted scrapie brain homogenate was incubated with an excessof healthy hamster brain homogenate. Samples were subjected to 0, 5, 10,20 or 40 cycles and the PrPres signal evaluated by immunoblot. Thelevels of PrPres increased exponentially with the number ofincubation/sonication cycles (FIG. 5). This result suggests thatincreasing the number of cycles could further diminish detection limits.

Example 4 Sonication Experiments in Blood Samples by Spiking withPrP^(Sc)

Heparinized rat blood was spiked with Scrapie hamster brain homogenateto reach a final dilution of 10:1. This mixture was incubated for 15 minat RT.

10 fold serial dilutions were made of this material using heparinizedrat blood. 50 μl of each dilution were centrifuged at 3,000 rpm for 10min. Plasma was separated from the pellet. 10 μl of plasma were mixed in50 μl of healthy hamster brain homogenate containing the PrP^(C)substrate for the conversion reaction. Samples were subjected to 11cycles of incubation-sonication. As a control same samples were mixed in50 μl of healthy hamster brain homogenate and kept at −20° C. untilneeded. 15 μl of sonicated and control samples were digested withproteinase K, separated by SDS-PAGE and analyzed by western blotting andPrP^(Sc) was detected as disclosed in the “Methods” section.

The results are reported in FIG. 6. These results show a clear increasein the detection of the protein after the amplification procedure, whichis especially evident at the lower concentration of PrP^(Sc) (forexample at the 1280 dilution). If we compare such results with thoseobtained on infected brain tissues, we have the confirmation that theamplification process works similarly in blood.

Example 5 High Throughput Cyclic Amplification

The use of a single-probe traditional sonicator imposes a problem forhandling many samples simultaneously, as a diagnostic test will require.We have adapted the cyclic amplification system to a 96-well formatmicroplate sonicator (Misonix 431MP- 20 kHz), which provides sonicationto all of the wells at the same time and can be programmed for automaticoperation. This improvement not only decreases processing time, but alsoprevents loss of material when compared to using a single probe. Crosscontamination is eliminated since there is no direct probe intrusioninto the sample. The latter is essential to handle infectious samplesand minimize false positive results. Twenty cycles of 1 h incubationfollowed by sonication pulses of 15 sec or 30 sec gave a significantamplification of PrPres signal, similar to that previously observedusing a traditional sonicator.

Example 6 The Factors Necessary for Amplification Are in aDetergent-Resistant Membrane Fraction

The subcellular location where the PrP conversion occurs during thedisease pathogenesis is not yet ascertained. However, both PrP^(C) andPrP^(Sc) have been reported to be located in a special region of theplasma membrane which is resistant to mild detergent treatment due tothe relatively high content of cholesterol and glycosphingolipids (Veyet al., 1996; Harmey et al., 1995). These membrane domains are namedlipid-rafts or detergent-resistant membranes (DRM) and are rich insignaling proteins, receptors and GPI-anchored proteins. We haveconfirmed that 100% of PrPC in brain is attached to this fraction, whichcontains <2% of the total proteins (FIG. 7). Thus, the simple step oflipid-raft isolation allows a dramatic enrichment in PrP^(C). Similarresults were obtained in the isolation of lipid-rafts from scrapie brainhomogenate, in which PrP^(Sc) was recovered in the rafts.

To evaluate whether the factors needed to amplify PrP are contained inlipid-rafts, we purified them from the brain of healthy animals andadded minute quantities of highly pure PrP^(Sc) extracted from the brainof sick animals. Amplification in lipid-rafts was equivalent to thatobtained with total brain extract (FIG. 8), since the amount of PrPresproduced after amplification was similar in both conditions. This resultindicates that all elements required for PrP conversion andamplification (including the so-called “Factor X”; (Telling et al.,1995)) are contained in this specialized membrane domain. Therefore,identification and isolation of the factors needed for PrP conversionshould be possible by further separation of proteins from thelipid-rafts and monitoring their activity by cyclic amplification. Inaddition, lipid-rafts constitute a possible replacement for the use oftotal brain homogenate in the cyclic amplification procedure as a sourceof PrP^(C) substrate and other endogenous factors implicated in theconversion.

Example 7 Pre-Symptomatic Diagnosis in Experimental Animals

To study the pre-symptomatic diagnosis of hamsters experimentallyinfected with scrapie, we screened 88 brain samples at different stagesduring the preclinical phase, half of which were non-infected controls.Brain was taken every week (4 per each group) and subjected to 20 cyclesof amplification. The results showed that the method is able to detectthe abnormal protein in the brain even at the second week afterinoculation, far before the animals develop any symptoms (FIG. 9).Without cyclic amplification, PrP^(Sc) was detected in the brain at weeksix post-infection, only 4 weeks before the appearance of the clinicaldisease. No amplification was detected in any of the control animalsthat were not infected with scrapie.

Example 8 Application of Cyclic Amplification to Human Brain Samples

To analyze the application of the cyclic amplification procedure tohuman samples from brain of people (cadavers) affected byCreutzfeldt-Jakob disease (CJD), we incubated brain homogenates ofseveral CJD patients (or normal controls) with healthy human brainhomogenate and carried out the cyclic amplification procedure. Theresults show that there was significant amplification in samples ofsporadic CJD brain analyzed and in none of the 4 control samples (FIG.10). Interestingly, amplification was obtained only in the samples thathad shown to be infectious and thus able to convert non-mutated PrP^(C),while it did not work when the mutant protein is not capable to convertthe wild type protein. These data support the conclusion that the methodworks in human samples similarly as shown before for animal samples.

Example 9 Diagnosis in Blood by Cyclic Amplification

Infectivity studies suggested that at least in experimental animalsPrP^(Sc) is present in blood in late-stage animals (Brown et al., 2001).In order to perform the blood detection of PrP^(Sc) by cyclicamplification, we preferred first to selectively concentrate the samplein the protein to be detected and to eliminate the bulk of very abundantblood proteins, such as albumin or hemoglobin. The following fourdifferent protocols have been shown effective for this purpose.

1. Preparation of Blood Cells Ghosts

Heparinized hamster blood was centrifuged at 2,500 rpm at 4° C. Theplasma and cellular fraction were separated and frozen at −80° C. untilneeded. 0.5 ml of blood cell package was washed 3 times in 12-15 vol offresh cold PBS, pH 7.6. The cells were resuspended in 12-15 vol of 20mOsM sodium phosphate buffer pH 7.6 and stirred gently for 20 min onice, then centrifuged at 30,000 rpm for 10 min at 4° C. The supernatantwas discarded, the pellet was washed 3 times in 20 mOsM sodium phosphatebuffer. The final pellet was resuspended in PBS containing 0.5% TritonX-100, 0.5% SDS and protease inhibitors. 15 μl of this suspension wasmixed v/v with 10% healthy hamster brain homogenate and subjected to 20cycles of incubation-sonication. 20 μl of sonicated and control sampleswere digested with proteinase K, separated by SDS-PAGE and analyzed bywestern blotting and PrP^(Sc) was detected as disclosed in the “Methods”section. The results show the detection of the PrP^(Sc) after theamplification procedure in the blood samples from infected animals (FIG.11). In the blood samples from non-infected animals there is no signalafter amplification. Without amplification is not possible to detect thepresence of PrP^(Sc) (FIG. 11).

2. Sarkosyl Extraction

Heparinized hamster blood was centrifuged at 2,500 rpm at 4° C. 0.5 mlof blood cell package was diluted (v/v) in 20% sarkosyl and incubatedfor 30 minutes. The sample was centrifuged in Beckman TL100ultracentrifuged at 85,000 rpm for 2 hrs at 4° C. The pellet was washedand resuspended in PBS containing 0.5% Triton X-100, 0.5% SDS andprotease inhibitors. 15 μl of this suspension was mixed v/v with 10%healthy hamster brain homogenate and subjected to 20 cycles ofincubation-sonication. 20 μl of sonicated and control samples weredigested with proteinase K, separated by SDS-PAGE and analyzed bywestern blotting and PrP^(Sc) was detected as disclosed in the “Methods”section. The results show the detection of the PrP^(Sc) after theamplification procedure in the blood samples from infected animals (FIG.12). In the blood samples from non-infected animals there is no signalafter amplification. Without amplification is not possible to detect thepresence of PrP^(Sc) (FIG. 12).

3. Lipid Raft Extraction

Heparinized hamster blood was centrifuged at 2,500 rpm at 4° C. 0.5 mlof blood cell package was diluted (v/v) in PBS with 1% Triton X-100 andincubated for 30 minutes at 4° C. The sample was diluted 1:2 in sucrose60% and placed in the bottom of a centrifuge tube. 7 ml of sucrose 35%were placed carefully over the sample. 1.5 ml of sucrose 15% was layeredin the top of the gradient. The tube was centrifuged at 150,000 rpm for18 hrs at 4° C. The lipid rafts were recovered washed in PBS andcentrifuged at 28,000 rpm during 1 hr at 4° C. The pellet was washed andresuspended in PBS containing 0.5% Triton X-100, 0.5% SDS and proteaseinhibitors. 15 μl of this suspension was mixed v/v with 10% healthyhamster brain homogenate and subjected to 20 cycles ofincubation-sonication. 20 μl of sonicated and control samples weredigested with proteinase K, separated by SDS-PAGE and analyzed bywestern blotting and PrP^(Sc) was detected as disclosed in the “Methods”section. The results show the detection of the PrP^(Sc) after theamplification procedure in the blood samples from infected animals (FIG.13). In the blood samples from non-infected animals there is no signalafter amplification. Without amplification is not possible to detect thepresence of PrP^(Sc) (FIG. 13).

4. Buffy Coat Preparation.

Heparinized hamster blood was centrifuged at 1,500 rpm at 4° C. for 10min. The buffy coat was carefully recovered using standard proceduresand kept at −80° C. until needed. The frozen buffy coat was resuspendedin PBS containing 0.5% Triton X-100, 0.5% SDS and protease inhibitors.15 μl of this suspension was mixed v/v with 10% healthy hamster brainhomogenate and subjected to 20 cycles of incubation-sonication. 20 μl ofsonicated and control samples were digested with proteinase K, separatedby SDS-PAGE and analyzed by western blotting and PrP^(Sc) was detectedas disclosed in the “Methods” section. The results show the detection ofthe PrP^(Sc) after the amplification procedure in the blood samples frominfected animals (FIG. 14). In the blood samples from non-infectedanimals there is no signal after amplification. Without amplification isnot possible to detect the presence of PrP^(Sc) (FIG. 14).

Methods Preparation of Brain Homogenates.

Brains from Syrian golden hamsters healthy or infected with the adaptedscrapie strain 263 K were obtained after decapitation and immediatelyfrozen in dry ice and kept at −80° C. until used. Brains werehomogenized in PBS and protease inhibitors (w/v) 10%. Detergents (0.5%Triton X-100, 0.05% SDS) were added and clarified with low speedcentrifugation (10,000 rpm) for 1 min.

Preparation of the Samples and Cyclic Amplification.

Serial dilutions of the scrapie brain homogenate were made directly inthe healthy brain homogenate. 30 μl of these dilutions were incubated at37° C. with agitation. Each hour a cycle of sonication (5 pulses of 1sec each) was done using a microsonicator with the needle immersed inthe sample. These cycles were repeated several times (5-20).

PrP^(Sc) Detection.

The samples were digested with PK 100 μg/mL for 90 min at 37° C. Thereaction was stopped with PMSF 50 mM. Samples were separated by SDS-PAGE(under denaturing conditions) and electroblotted into nitrocellulosemembrane in CAPS or tris-glycine transfer buffer with 10% methanolduring 45 min at 400 mA. Reversible total protein staining was performedbefore blocking of the membrane with 5% non-fat milk. Thereafter, themembrane was incubated for 2 hr with the monoclonal antibody 3F4(1:50,000). Four washes of 5 min each were performed with PBS, 0.3%Tween20 before the incubation with the horseradish peroxidase labelledsecondary anti-mouse antibody (1:5000) for 1 hr. After washing, thereactivity in the membrane was developed with ECL chemiluminescence Kit(Amersham) according to manufacturer's instructions.

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1. A method for the detection of a conformational disease which ischaracterized by a conformational transition of Prion Protein betweenthe PrP^(C) non-pathogenic conformer and the PrP^(SC) pathogenicconformer, by assaying a marker of said disease within a sample, whichmethod comprises: (i) contacting said sample with an amount of thePrP^(C) non-pathogenic conformer; (ia) incubating said sample with saidPrP^(C) non-pathogenic conformer; (ii) disaggregating any aggregateseventually formed during step (i); and (iii) determining the presenceand/or amount of said PrP^(SC) pathogenic conformer within the sample,the pathogenic conformer being a marker for the presence of saiddisease, wherein steps (ia) and (ii) form a cycle which is repeated atleast twice before carrying out step (iii).
 2. The method of claim 1,wherein the cycle is repeated from 5 to 40 times before carrying outstep (iii).
 3. The method of claim 1, wherein step (i) is carried outunder physiological conditions.
 4. The method of claim 1, wherein theamount of the PrP^(C) non-pathogenic conformer in step (i) is an excessamount.
 5. The method of claim 1, wherein the conformational disease isa transmissible conformational disease.
 6. The method of any one of thepreceding claims, wherein the sample to be analysed is subjected to apre-treatment for selectively concentrating the PrP^(SC) pathogenicconformer in the sample.
 7. The method of claim 6, wherein thepre-treatment is the extraction from the sample of a fraction which isinsoluble in mild detergents.
 8. An assay for a marker of aconformational disease which is characterized by a conformationaltransition of Prion Protein between the PrP^(C) non-pathogenic conformerand the PrP^(SC) pathogenic conformer, within a sample, which assaycomprises the following steps: (i) contacting said sample with an amountof the PrP^(C) non-pathogenic conformer; (ia) incubating said samplewith said PrP^(C) non-pathogenic conformer; (ii) disaggregating anyaggregates eventually formed during step (i); and (iii) determining thepresence and/or amount of said PrP^(SC) pathogenic conformer within thesample, the PrP^(SC) pathogenic conformer being a marker for thepresence of said disease, wherein steps (ia) and (ii) form a cycle whichis repeated at least twice before carrying out step (iii).
 9. A kit foruse in the assay of claim 8 which comprises a known amount of thenon-pathogenic conformer, a multi-well microtitre plate and a multi-wellsonicator.
 10. A method for identifying a compound which modulates theconformational transition of Prion Protein between the PrP^(C)non-pathogenic conformer and the PrP^(SC) pathogenic conformer,comprising: (i) contacting an amount of the PrP^(C) non-pathogenicconformer with an amount of the PrP^(SC) pathogenic conformer (a) in thepresence of said compound and (b) in the absence of said compound; (ii)disaggregating any aggregates eventually formed during step (i); and(iii) determining the amount of the PrP^(SC) pathogenic conformer (a) inthe presence of said compound and (b) in the absence of said compound.11. A method for detecting the presence of a PrP^(SC) pathogenic form ofPrion Protein within a sample, comprising: (i) contacting the samplewith an amount of the PrP^(C) non-pathogenic prion protein; (ia)incubating the sample with the PrP^(C) non-pathogenic prion protein;(ii) disaggregating any aggregates formed during step (ia); repeatingsteps (ia)-(ii) two or more times; and then (iii) determining thepresence and/or amount of PrP^(SC) pathogenic prion protein within thesample.
 12. Apparatus for use in the method of any one of claims 1 and2-7 or for use in the assay of claim 8, comprising a microtitre plate,multi-well sonicator and an amount of the PrP^(C) non-pathogenicconformer.